Department of Physiology and Cell Physiology of Alpine Plants, University of Innsbruck, Institute of Botany, Sternwartestrasse 15, 6020 Innsbruck, Austria.
Protoplasma. 2010 Jul;243(1-4):63-71. doi: 10.1007/s00709-009-0053-8. Epub 2009 Jun 3.
Leaflets of Sphagnum capillifolium were exposed to temperatures from -5 degrees C to +60 degrees C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at -1.1 degrees C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 microm indicates a cell volume reduction of approximately -82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (-1.1 degrees C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (-16.1 degrees C; LT(50)) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5 degrees C which is significantly below the heat tolerance of chlorophyllous cells (49.9 degrees C; LT(50)). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.
将金发藓叶片暴露在显微镜载物台上，在受控条件下将温度从-5°C 调节至+60°C。使用光学显微镜和荧光显微镜成功监测到这些温度处理的细胞反应。除了观察到叶片在冷冻时的细胞质外渗和热暴露时的细胞质外渗的可观察细胞变化外，还使用微光纤和叶绿素荧光计研究了与之伴随的破坏光合系统 II (PS II) 的临界温度阈值，该叶绿素荧光计安装在显微镜载物台上。在测量过程中，当叶片浸入水中时，在 -1.1°C 时，金发藓的叶绿素细胞立即出现扩展的冷冻质壁分离。细胞收缩导致的可见的冷冻质壁分离是高度动态的，在 2 秒内完成。在冷冻质壁分离过程中，叶绿体的平均投影直径从 8.9μm 减少到 3.8μm，表明细胞体积减少了约 -82%。即使通过叶片样本浸入的冷冻水中，也可以同时测量 PS II 的叶绿素荧光。冷冻质壁分离伴随着基本叶绿素荧光的突然增加。PS II 的临界冷冻温度阈值与冰核形成温度（-1.1°C）相同。这明显高于金发藓叶片发生霜害的温度阈值（-16.1°C；LT(50)），比欧洲阿尔卑斯山夏季大多数高等植物观察到的温度阈值高。PS II 的高温阈值为 44.5°C，远低于叶绿素细胞的耐热性（49.9°C；LT(50)）。研究结果表明，光和荧光显微镜技术结合同时进行叶绿素荧光测量，可能成为一种有用的工具，用于研究对完整叶片组织的细胞结构和初级光合作用过程的热、低温和冰包埋效应。
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